|Keywords (Extracted from title, table of contents and abstract of thesis)
soluble antigens, particulate antigens, humoral immune responses, proliferative immune responses, rana tigrina, humoral immunological memory, frog, human gamma globulin
The present work was carried out to study the humoral immunological memory in the frog, Rana tigrina using adult and tadpoles. The antigens used for immunization were in a soluble and particulate form. Human gamma globulin (HGG) is a soluble antigen while Salmonella typhi (s. typhi) in particulate form. Moreover, proliferative responses were also studied by observing histological sections of spleen. To determine the effective route of immunization a pilot study was carried out on adult frogs by using particulate antigens, S. typhi. In this study various routes were used such as intraperitoneal (ip), intramuscular (im) and via dorsal lymph sac. Antigen was administered both in saline as well as in adjuvant (Freund's complete adjuvant). In either case it was found that dorsal lymph sac route immunized the animals better than that of intramuscular or intraperitoneal, when treated two, four and six weeks after immunization. In further studies dorsal lymph sac (dls) route was used in adult frogs. In tadpoles, however, intraperitoneal route was used, as it was difficult to administer antigen via dorsal lymph sac being absent or poorly developed.
In adult frogs soluble antigen HGG was used to determine humoral anti HGG antibodies following immunization at day 0 (primary treatment) and then four weeks after (challenge) and tested for antibodies two, four and six weeks after the challenge. It was found that those frogs, which received only one dose of HGG either as primary treatment or as a challenge, produced low antibody titres when compared with those frogs, which received HGG both in primary treatment as well as in the challenge. Moreover, use of adjuvant had not significant enhancing affect, rather it decreased the antibody titres when compared with that of the use of antigen in saline. Studies on humoral antibody production using particulate antigens, S. typhi showed similar results. The use of S. typhi in both primaries as well as in challenge produced higher anti-S. typhi antibodies as compared to that of the frogs, which received S. typhi either during primary or in the challenge. Moreover, in this case the use of adjuvant had enhancing effect.
The use of antigen HGG or S. typhi for immunizing tadpoles (stage 50) also showed some noticeable results. When tadpoles were primed during stage 50 and were allowed to metamorphose and tested four weeks after, the froglets showed no significant anti-HGG or anti-S. typhi antibodies. A batch of tadpoles were primed during stage 50 with an antigen, were then allowed to metamorphose. Four weeks after metamorphosis the froglets received challenge of the antigen in saline or in adjuvant, mounted good antibody titres when compared to control, which had received no antigen in larval conditions but only the challenge as froglets. This indicates that immunization during larval conditions primed the animals and this persisted even during metamorphosis. In these studies the use of adjuvant affected antibody production positively.
The study of pyrininophilic responses were carried out by immunizing frogs or tadpoles, then three weeks after the challenge spleens were removed for phyrininophilia and studied by following methyl green pyronin staining. The forgs, which received antigens (HGG or S. typhi) only in single dose (primary treatment or the challenge) showed less pyriminophilia as pyrininophilic cells were uniformly distributed throughout the spleen. The frogs, which received antigen in both primary as well as in challenge, produced intensive pyrininophilic response. It can be seen as heavy clusters or clumps of cells in white pulp and on the peripheral regions of the spleen. When antigen was administered during larval stages only the metamorphosing froglets exhibited little or no pyrininophilia in their spleens. On the other hand, those froglets, which received antigen both in larval as well as in the challenge (after metamorphosis) produced very high splenic proliferation. Same results have been obtained by cytospin smear method.
In these studies it was found that peak time for proliferative responses is 3 weeks after the challenge while for humoral antibody production is 4 weeks after the challenge